Mammalian transient receptor potential (TRP) channels are described as six-transmembrane (6-TM) cation-permeable channels. TRP channels control the gating of voltage-dependent Ca2+, K+, and Cl−, and are characterized as calcium-permeable channels with polymodal activation properties. TRP protein structure is thought to be a channel forming structure composed of six transmembrane (TM) domains with a pore domain (P) located between the fifth (S5) and sixth (S6) TMs. TRP channels are activated by three major mechanisms; receptor, ligand and environment direct activation. Receptor activation is carried out by G protein coupled receptors (GCPRs) and tyrosine kinases in three modes which result in liberation of Ca2+ from intracellular stores: hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP2), diacylglycerol (DAG) and inositol (1,4,5) triphosphate (IP3). Ligand activation occurs by exogenous small molecules (capsaicin, icilin, 2-APB), endogenous lipids or metabolism products (diacylglycerols), purine nucleotides and metabolites (ADP-ribose), and inorganic ions. TRP channels are also activated by environmental triggers such as ambient temperature.
The TRPC subfamily was established by the identification of the first mammalian TRP, TRPC1. Common TRPC motifs are composed of 2-3 ankyrin-like domain repeats and a coiled-coil domain in the N-terminal, followed by six transmembrane domains, the C-term TRP box and a calmodulin (CaM) binding site. The CaM domain in TRPC4 is a calcium binding domain which resembles the CaM protein, which is normally small (˜140+ amino acid in length) dumbbell-shaped composed of two structurally similar globular domains separated by a flexible hinge central helix. The globular domains are homologous and contain pairs of Ca2+ binding helix-loop-helix motifs which are referred to as the EF hand motifs. The typical mechanism of calcium binding occurs at these EF hands, which are composed of two α-helices linked to a 12-residue loop. The EF hand domains become exposed to effectors and targets by protein conformational change. The exposed hydrophobic regions in turn bind basic amphiphilic helices (BAA helices). The hinge of CaM allows for the proteins harboring a CaM domain to contact and activate targets (FIG. 1). CaM is highly conserved in animals and plants and acts on many targets including ion channels.
TRPC5 is expressed homomerically and also heteromerically complexes with TRPC4. TRPC4 and TRPC5 are highly homologous, and are highly expressed in the human brain, uterus, ovary and kidney cells. TRPC4 is a nonselective cation channel which is uniquely activated by Gq/11 family GPCRs through activation of PLCβ, and receptor kinases and receptor tyrosine kinases. Although studies using TRPC4 have shown that activation requires phospholipase C (PLC) activity, neither IP3 nor DAG is sufficient to activate TRPC4. TRPC4 contains a PDZ-binding motif. PDZ domains are common structural motifs which aid proteins in signaling and anchoring transmembrane proteins to the cytoskeleton. PDZ domain scaffolding proteins, as well as signaling molecules, co-immunoprecipitate with TRPC4. PDZ domain is a common structural domain of 80-90 amino-acids found in the signaling proteins of bacteria, yeast, plants, viruses and animals. PDZ is an acronym combining the first letters of three proteins—post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1)—which were first discovered to share the domain. PDZ domains are also referred to as DHR (Dlg homologous region) or GLGF (glycine-leucine-glycine-phenylalanine) domains. These domains help anchor transmembrane proteins to the cytoskeleton and hold together signaling complexes.
Almost every cell type scrutinized contains at least one TRP channel. This large family of physiological important channels has been implicated in many human diseases. Most of the TRP channels are conserved in mice, rats, and humans. Knockout mice studies have proven to be insightful for determining TRP channel functions. Trpc2 deficient mice are unable to distinguish male from female counterparts and TRPV6 is upregulated in prostate cancer. TRPC4 transcripts and protein are expressed in primary cultured mouse vascular endothelial cells (MAECs) and the channels can be activated by store-depleted protocols in MAECs. In Trpc4 deficient mice, agonist induced Ca2+ entry is significantly reduced. Trpc4−/− mice exhibit significant decrease in endothelium-dependent vasorelaxation in the blood vessels. The Trpc4 deficient mice display decreased microvascular permeability, and have altered GABA transmitter release from thalamic interneurons. Although TRPC4 is expressed in the nervous system it has not been validated previously as a target for neuropathic pain and there were no known specific inhibitors for the channel.
The human TRPC4 protein contains multiple ankyrin domains throughout and within the N-terminus along with a coiled-coil domain. The N-terminus of TRPC4 is very important for subunit assembly and pore formation. Two regions in the N-terminus are essential for channel assembly in TRPC channels and more specifically TRCP4; the third and fourth ankyrin repeats and the region downstream the coiled-coil domain. The second and third ankyrin repeats are represented by F59-S137 in TRPC4. Both of these domains are able to self associate but have not been shown to interact with one another. The last 18 amino acids of the region downstream of the coiled-coil domain are represented in TRPC4 by 287-A RLKLAIKYRQKEFVAP-304, and in TRPC6 by 363-SRLKLAIK YEVKKFVAHP-380. These peptides have been identified to be involved in channel assembly of TRPCs and more specifically TRPC4. There are two domains in the TRPC4 protein that are responsible for oligomerization. The first domain contains the N-term ankyrin repeats and the coiled-coil domain (M1-P304) and the second domain corresponds to the putative pore region and the C-terminal tail (I516-L974). Two models exist in which the TRPC4 channel becomes functional upon subunit assembly. One model is that the third ankyrin repeat initiates a molecular zippering process. In this model each interacting domain would have the ability to tetramerize. In another model, the first interaction domain forms a dimer between two subunits and the second domain is responsible for the formation of a dimer between two other subunits. The N-terminal of both TRPC4 and TRPC5 including at least the first ankyrin repeat are essential for both homo and hetero-subunit assembly. TRPC4 protein homo and heteromeric pore formation is critical for protein function; therefore, agents that block TRPC4 multimeric formation are reasonable candidates for TRPC4 protein inhibitors.
Neuropathic pain is a chronic disease resulting from a dysfunction in the nervous system. This nervous system dysfunction often occurs due to peripheral nerve injury concentrated at the dorsal root ganglia (DRG), sensory neurons. Abnormal nervous function arises from injured axons, and from intact nociceptors that share receptivity with the injured nerve. The pathological conditions include prolonged hyperalgesia, allodynia, and loss of sensory function. The classical presentation of neuropathic pain includes ubiquitous pain not otherwise explainable, sensory defects, burning pain, pain to light on the skin and sudden pain attacks without a clear provocation. Inflammation and trauma are major causes of nerve injuries. The genetic disorders causing distorted connectivity, structure or survival of neurons may also result in neuropathic pain.